Map-based Cloning And Functional Analysis Of The 824ys Mutant Gene Controlling Yellow-green Leaf In Rice

Chlorophyll (Chl) is the main component of the photosynthetic pigments. Chl molecules universally exist in photosynthetic organisms and perform essential processes of harvesting light energy in the antenna systems and by driving electron transfer in the reaction centers. In higher plants, there are two Chl species, Chl a and Chl b. The photosynthetic reaction centers contain only Chl a, and the peripheral light-harvesting antenna complexes contain Chl a and Chl b. Chl a is synthesized from glutamyl-tRNA, and Chl b is synthesized from Chl a at the last step of Chl biosynthesis. So far, genes for all 15 steps in the Chl biosynthetic pathway have been identified in higher plants, at least in angiosperms represented by Arabidopsis thaliana. Analysis of the complete genome of Arabidopsis showed that it has 15 enzymes encoded by 27 genes for Chl biosynthesis from glutamyl-tRNA to Chl b. However, only six genes encoding three enzymes involved in Chl biosynthesis have been identified in rice(Oryza sativa), including OsCHLH, OsCHLD and OsCHLI genes for three subunits of Mg-chelatase, YGL1 gene for Chi synthase, OsCAOl and OsCAO2 genes for Chl a oxygenase.According to the number of vinyl side chains, Chls of oxygenic photosynthetic organisms are classified into two groups:3,8-divinyl Chi (DV-Chl) and 3-vinyl Chl (monovinyl Chl, MV-Chl). Almost all of the oxygenic photosynthetic organisms contain MV-Chls, regardless of the variation in their indigenous environments.3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll synthesis. So far, three 8-vinyl reductase genes (DVR, bciA and slr 1923) have been characterized from Arabidopsis thaliana, Chlorobium tepidum and Synechocystis sp. PCC6803. Enzymatic assay confirmed that recombinant DVR and BciA proteins can catalyze the conversion of DV-Chlide a and DV-Pchlide a to corresponding MV-compounds, respectively. However, no divinyl reductase gene has yet been identified in monocotyledonous plants.In this study, a spontaneous mutant '824ys' was isolated from indica rice'824B'. The mutant exhibited a yellow-green leaf phenotype throughout the growth stage, and grew very slowly. Although its days to heading increased 16 d, but its height of plants and number of productive tillers decreased by 25.6%and 64.9%compared with the wild type, respectively. Its contents of Chl a, b and total Chls were 41 to 48%,9 to 22%and 33 to 42%of that from wild-type, respectively, indicating that the mutant phenotype resulted from reduced Chl level. Transmission electron microscopic analysis revealed that the shape of 824ys mutant chloroplasts was irregular compared with the wild-type chloroplasts. The thylakoid membranes of the mutant chloroplasts were disrupted. The grana stacks in the mutant appeared less dense. These results indicated that the development of chloroplast thylakoids was suppressed in the mutant.Genetic analysis suggested that the yellow-green leaf phenotype in the 824ys mutant is controlled by a single recessive nuclear gene. This mutant gene was preliminarily mapped on the short arm of rice chromosome 3,5.2 cM from SSR marker RM282. Subsequently, by using F2 population from the cross between the 824ys mutant with a japonica cultivar '02428', the 824ys locus was further mapped between the InDel marker PR809 and PR826, at genetic distances of 0.2 and 0.4 cM, respectively. The two markers mapped a 142 kb DNA region on two bacterial artificial chromosomes. In the corresponding region, one open reading frame (ORF) (Os03g22780) was found encoding a putative isoflavone reductase, and sequence BLASTX showed it has a significant similarity to 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR,65%identities) in Arabidopsis. Then, we cloned the Os03g22780 gene from the 824ys mutant and the corresponding wild-type parent 824B. DNA sequencing results revealed that nine base pairs were deleted at nucleotide 952 to 960 in the ORF in the 824ys mutant, which resulted in the deletion of three amino acid residues (Lys, Val and Pro). Therefore, the Os03g22780 gene was considered as the candidate gene of 824ys, and designated tentatively as OsDVR gene. BLAST search in the rice genome database revealed that OsDVR is a single-copy gene with an ORF of 1218 bp and encodes a 405-amino acid protein with the molecular mass of approximately 43 kD. OsDVR protein contains an apparent chloroplast-targeting sequence of 58 amino acid residues at its N terminus.Chl compositions of the 824ys mutant were examined by high-performance liquid chromatography (HPLC). The result showed that the mutant accumulated only DV-Chls, instead of MV-Chls, so the mutant has defective divinyl reductase (DVR). Accordingly we concluded the candidate gene underlying the 824ys mutant phenotype is OsDVR. To test the DVR activity of the recombinant OsDVR protein, the recombinant OsDVR and Osdvr proteins were expressed in E. coli, and four DV-tetrapyrroles (DV-Chl a,-Chi b,-Chlide a and-Chlide b) were used as substrates. The cell extracts were incubated with the four different DV-tetrapyrroles, respectively, in the presence of NADPH. The results demonstrated that the recombinant OsDVR protein not only converted DV-Chlide a to MV-Chlide a, but also converted DV-Chl a to MV-Chl a, which confirmed that the OsDVR (Os03g22780) encodes a functional divinyl reductase in rice, and that the deletion of three amino acid residues (Lys, Val and Pro) results in inactivation of OsDVR in the 824ys mutant.Based upon these results, we firstly succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed a novel DVR activity to convert divinyl chlorophyll a to mono vinyl chlorophyll a.